A draft of the human genome has been published; however, much of the data comprise a rough draft, which is that the sequences are not continuous resulting in gaps in the sequence data. There is an urgency in filling the innumerable sequence gaps of the human genome resulting from inherent shortcomings of the sequencing strategies employed. The proposed research is a novel technique to target sequencing to a specific region of interest in the genome (e.g., sequence gap) that is accomplished without the need to clone. The goal of the project is to develop and demonstrate that OmniPlex technology has the capability of chromosome walking from a known starting point into unknown regions of a gene allowing targeted sequencing of a specific region, filling gaps in sequence data. The first step, Phase I, will be the development of a chromosome walking approach and the of feasibility of this approach in filling both captured and uncaptured sequence gaps. There is an immediate need for a universal technology applicable to sequencing gaps that are in regions that are uncaptured by clones as well as other difficult regions that are clonable would be of greatest value. The novel chromosome technology envisioned is a technological innovation that will provide a more efficient (cost and time) and universal approach to effectively sequencing across all types of gaps in mammalian genomes as OmniPlex is a method of preparing genomic DNA for sequencing without the need to clone in host systems. The specific aims of the proposed research are: 1) develop a robust method to amplify an OmniPlex library to walk into unknown genomic regions in a mammalian genome, 2) use OmniPlex walking to sequence gaps not captured in BACs in human Chromosome 22, and 3) use OmniPlex walking to sequence 10 gaps captured in BACs in the mouse or human genome. Phase II will consist of the development and validation of the directed sequencing technology of OmniPlex in broader commercial application, as well as a tool for large-scale genome sequencing centers. OmniPlex chromosome walking will provide a method to direct sequencing to targeted regions of the genome in a cost-efficient and rapid manner, which current technology (PCR and shotgun sequencing) do not provide.